KR100449474B1 - Production method of polyester containing epoxy group in side chain and production method of crosslinked polymer - Google Patents

Abstract

Provided is a PHA having improved physicochemical properties, and a method for biosynthesizing PHA having an epoxy group at a side chain end. Specifically, a method for producing a polyester containing an epoxy group in a side chain using 1-alkene as a raw material, wherein the 1-alkene is brought into contact with a microorganism having the ability to introduce the 1-alkene and convert it to the polyester. The manufacturing method of the polyester containing the epoxy group in the side chain which has a process of converting into this polyester is provided. Further, there is provided a method for producing a crosslinked polymer comprising reacting a polyester obtained by the method described above with a diamine compound.

Description

TECHNICAL METHOD OF POLYESTER CONTAINING EPOXY GROUP IN SIDE CHAIN AND PRODUCTION METHOD OF CROSSLINKED POLYMER}

The present invention relates to a method for producing polyester microorganisms.

To date, many microorganisms have been reported to produce poly (3-hydroxybutyric acid) (hereinafter abbreviated as PHB) or other polyhydroxyalkanoate (PHA) and accumulate in cells ("Biodegradable Plastic Handbook"). ", Biodegradable Plastics Research Society, NTS CO., Ltd., P178-197, 1995). These polymers can be used for production of various products by melt processing and the like as conventional plastics. In addition, since they are biodegradable, they have the advantage of being completely decomposed by microorganisms in nature, and do not cause contamination by remaining in the natural environment like many synthetic polymer compounds in the past, and incineration treatment is unnecessary, so dioxin There is no fear of generating harmful substances such as environmental hormones. In addition, it is also excellent in biocompatibility and is also expected to be applied as a medical soft member or the like (Japanese Patent Laid-Open No. 5-159).

In recent years, in the industrial use of such PHAs, attempts have been made to produce PHAs composed of units different from those of ordinary monomer units and to widen the variety of physical and chemical properties.

As one of these methods, attempts have been made to improve the physicochemical properties of PHA by introducing an epoxy group into the side chain of PHA and performing crosslinking reaction or chemical modification with this portion as an active point.

Macromolecules, 31, P1480-1486 (1998) and Journal of Polymer Science: Part A: Polymer Chemistry, 36, P2381-2387 (1998), added Pseudomonas oleovorans in different ratios of sodium octanoate and unsaturated fatty acid 10-undecenoic acid. By culturing in a medium, a PHA comprising various proportions of units having an unsaturated bond at the end of the side chain is synthesized, and then the unsaturated portion is chemically epoxidized with 3-chlorobenzoic acid to contain an epoxy group at the end of the side chain. Is reported to synthesize PHA. Furthermore, in Journal of Polymer Science: Part A: Polymer Chemistry, 36, P2389-2396 (1998), crosslinking is carried out using 2-ethyl-4-methylimidazole as the polymerization initiator as the epoxy PHA and using amber acid anhydride. It is reported that reaction was performed.

Thus, in improving the physicochemical properties of PHA, the epoxy group at the side chain end is very useful, but there is no synthesis method other than chemically epoxidizing the unsaturated portion at the side chain end, and the operation is very complicated and the cost problem is a real problem. Even there is no advantage.

It is therefore an object of the present invention to provide a method for solving the above problems.

1 is a diagram showing 1 H-NMR of a polymer obtained in Example 1;

FIG. 2 is a diagram showing 1 H-NMR of a polymer obtained in Example 2. FIG.

3 is a diagram showing 1 H-NMR of a polymer obtained in Example 3. FIG.

4 shows 1 H-NMR of the polymer obtained in Example 4;

FIG. 5 is a diagram showing 1 H-NMR of the polymer obtained in Example 5. FIG.

FIG. 6 is a diagram showing 1 H-NMR of a polymer obtained in Example 6. FIG.

FIG. 7 shows the polymer production pathways from 1-alkenes of YN2 strains. FIG.

8 (a), 8 (b) and 8 (c) show the GC charts of the results displayed in Example 7;

9 (a), 9 (b) and 9 (c) show the GC charts of the results displayed in Example 8;

10 is a diagram showing a DSC chart of a polymer represented in Example 9;

11 (a) and 11 (b) show FT-IR charts of the polymers shown in Example 9. FIG.

According to a first aspect of the present invention, a method for producing a polyester containing an epoxy group in a side chain using 1-alkene as a raw material, wherein the microorganism having the ability to introduce the 1-alkene and convert it into this polyester It provides a process for producing a polyester comprising an epoxy group in the side chain characterized by having a step of contacting the alkene to convert to a polyester containing an epoxy group in the side chain.

In the present invention, it is preferable to include the step of culturing the microorganism in a medium containing 1-alkene.

Moreover, in this invention, it is preferable to further include the process of isolating the polyester produced by this microorganism.

Moreover, in this invention, it is more preferable that the said isolation | separation process includes the process of collect | recovering this polyester from this microbial cell.

According to a second aspect of the present invention, there is provided a method for producing a crosslinked polymer, comprising reacting a polyester obtained by the above method with a diamine compound.

[Detailed Description of Preferred Embodiments]

The polyester obtained according to the method of the present invention is formulated in the monomer unit (1):

(n: integer of 1 to 7)

The unit represented by contains at least 1 mol%.

In addition, the polyester obtained according to the method of the present invention is formulated in the monomer unit (2):

(m: integer of 1 to 7)

The unit represented by may contain at least 1 mol% more.

In addition, the polyester obtained according to the method of the present invention is formulated in the monomer unit (3):

(k: integer from 0 to 8)

At least 1 mol% of the unit represented by this may be contained further.

The 1-alkene which is a raw material in the method of the present invention is a 1-alkene having 7 to 12 carbon atoms, that is, 1-heptene, 1-octene, 1-nonene, 1-decene, 1-undecene, 1-dodecene It is preferable to use.

In addition, the number average molecular weight of the polyester obtained by the present invention is 10,000 to 1,000,000, more specifically 10,000 to 500,000.

(microbe)

Microorganisms used in the method of the present invention include the ability to epoxidize 1-alkenes and convert them to the corresponding epoxy alkanes; The ability to convert the terminal of this epoxy alkane compound into epoxidized carboxylic acid; A microorganism having the ability to convert this epoxidized carboxylic acid to polyester, and such microorganisms include microorganisms belonging to the genus Pseudomonas, and more specifically, Pseudomonas chi Korea used in the examples of the present invention described below is YN2. Note (Pseudomonas cichorii YN2; FERM BP-7375).

Pseudomonas cichorii YN2 (FERM BP-7375), which is a microorganism used in the present invention, is a microorganism having the following properties, and has been placed in the International Patent Biological Depository of the National Association of Advanced Industrial Science and Technology (AIST). Deposited (Accession Number: FERM BP-7375).

Here, the mycological properties of YN2 strains are shown below.

(1) morphological properties

Incubation temperature: 30 ℃

Cell form: Bacillus (0.8 ㎛ x 1.5 to 2.0 ㎛)

Gram dye: Voice

Sporulation: Voice

Mobility: Positive

Colony shape: round; Full edge smoothing; Low convexity; Surface smoothing; Polish; Translucent

(2) physiological properties

Catalase: Positive

Oxidase: Positive

0 / F Test: Non Fermentation

Nitrate Reduction: Negative

Indole production: positivity

Glucose Oxidation: Negative

Arginine dihydrolase: voice

Urease: negative

Escrine hydrolysis: Negative

Gelatin hydrolysis: Negative

β-galactosidase: negative

Fluorescent Production in King's B Agar:

Growth at 4% NaCl: Positive (about)

Accumulation of poly-P-hydroxybutyric acid: Negative (＊)

Hydrolysis of Tween 80: Positive

* Determination of nutrient agar cultured colonies by Sudan black

(3) substrate magnetization ability

Glucose: Positive

L-Arabinose:

D-Mannose: Voice

D-mannitol: voice

N-acetyl-D-glucosamine: negative

Maltose: Voice

Potassium Gluconate: Positive

n-caprylic acid: positive

Adipic acid: Negative

dl-Nungumsan: By Yang

Sodium Citrate: Positive

Phenyl Acetate: Positive

This strain is also a microorganism disclosed in Japanese Patent Application No. 11-371863. This strain has the ability to epoxidize 1-alkenes to the corresponding epoxy alkanes, as shown in the following examples. Usually, the enzyme for exerting this ability is an alkenmonooxygenase. It is very likely that this strain also contains this alkenmonooxygenase. In addition, the discovery that this strain produces the corresponding epoxy alkanoic acid from alkenic acid has not been obtained. From the above result, it is suggested that the path | route which this strain produces the polyester shown by this invention is a path | route as shown in FIG.

(Cultivation process)

The medium used in the present invention may be any medium as long as it is an inorganic salt medium containing a nitrogen source such as phosphate, ammonium salt or nitrate. However, the productivity of PHA can be improved by adjusting the concentration of the nitrogen source. Since 1-alkene to be added is poor in solubility in water and has high volatility, it is necessary to supply it in gaseous state at the time of culturing and to keep it in a closed state while securing oxygen required by this microorganism.

As an example of an inorganic salt medium, the composition of the medium used for one Embodiment of the method of this invention is shown below.

(M9 badge)

Na ₂ HPO ₄ : 6.3

KH ₂ PO ₄ : 3.0

NH ₄ Cl: 1.0

NaCl: 0.5 g / L, pH = 7.0

(1 / 10N-M9 badge)

Na ₂ HPO ₄ : 6.3

KH ₂ PO ₄ : 3.0

NH ₄ Cl: 0.1

NaCl: 0.5 g / L, pH = 7.0

In addition, for the good growth and production of PHA, it is necessary to add about 0.3% (v / v) of the minor component solution shown below to the inorganic salt medium.

(Trace component solution)

Nitric triacetic acid: 1.5; MgSO ₄ : 3.0;

MnSO ₄ : 0.5; NaCl: 1.0;

FeSO ₄ : 0.1; CaCl ₂ : 0.1;

CoCl ₂ : 0.1; ZnSO ₄ : 0.1;

CuSO ₄ : 0.1; AlK (SO ₄ ) ₂ : 0.1;

H ₃ BO ₃ : 0.1; Na ₂ MoO ₄ : 0.1;

NiCl ₂ : 0.1 (unit: g / L)

As culture temperature, what is necessary is just the temperature which the said strain can proliferate favorably, and about 20 degreeC-about 30 degreeC are suitable.

The culture method may be any culture method as long as this microorganism grows and produces PHA, such as a liquid culture method and a solid culture method. In addition, the culture type includes, but is not limited to, batch culture, feed batch culture, semi-continuous culture, continuous culture, and the like.

The method conventionally performed can be applied to the acquisition of PHA from a culture comprising the cultured cell and the culture medium according to the present invention. When PHA is secreted into a culture medium, an extraction and purification method from the culture medium is used. When PHA is accumulated in the cell, an extraction and purification method from the cells is used. For example, the extraction of PHA from microorganism culture cells is the simplest and most commonly performed chloroform extraction, but in environments where organic solvents are difficult to use, surfactant treatment such as SDS, enzyme treatment such as lysozyme, EDTA, and char It is also possible to employ a method of recovering only PHA by removing components in cells other than PHA by pharmaceutical treatment such as sodium chlorate and ammonia.

In addition, Appl. Environ. Microbiol., 54, P2924-2932 (1998) reported the production of polyesters according to the process of the invention using Pseudomonas oleoborans, but the polyesters produced do not have epoxy groups in the side chains and at the ends of the side chains. Polyesters comprising units having double bonds and units whose side chains are saturated alkylene chains.

The polymer obtained by the method of the present invention can perform a chemical conversion similar to that of a polymer having an ordinary epoxy group. Specifically, the crosslinking reaction by hexamethylenediamine, amber acid anhydride, 2-ethyl-4-methylimidazole, or electron beam irradiation is mentioned. It is also possible to convert to a hydroxyl group or to introduce a sulfone group. It is also possible to add compounds with thiols or amines.

The present invention also provides a method for producing a crosslinked polymer obtained by reacting the polyester and a diamine compound. In more detail, the manufacturing method of the said crosslinked polymer obtained by making the said polyester and hexamethylenediamine react is provided. Such a reaction path proceeds along the reaction path as shown in the following scheme to produce a crosslinked polymer.

The reaction temperature is preferably 50 ° C to 120 ° C, and the reaction time is preferably in the range of 10 minutes to 120 minutes.

<Example>

Although an Example is shown below, this invention is not restrict | limited at all by the following Example.

(Example 1)

Colonies of YN2 strains on M9 agar medium containing 0.1% yeast extract were suspended in sterile saline solution such that OD (600 nm) = 1.0. The suspension was applied to 20 sheets of 1 / 10N-M9 agar containing no carbon source, and left incubated at 30 ° C in a 1-heptene atmosphere.

After 4 days, the cells were collected, washed with methanol, collected by centrifugation, and dried under reduced pressure.

50 mL of chloroform was added to the dry cells, and the mixture was stirred at 30 ° C. for 48 hours to extract PHA. The chloroform layer was filtered, concentrated by evaporator, added to cold methanol, and the precipitate was recovered and dried under reduced pressure.

(Example 2)

Production experiments and evaluations were carried out in the same manner as in Example 1 except that 1-heptene was changed to 1-octene.

(Example 3)

Production experiments and evaluations were carried out in the same manner as in Example 1 except that 1-heptene was changed to 1-nonene.

(Example 4)

Production experiments and evaluations were carried out in the same manner as in Example 1 except that 1-heptene was changed to 1-decene.

(Example 5)

Production experiments and evaluations were carried out in the same manner as in Example 1 except that 1-heptene was changed to 1-undecene.

(Example 6)

Production experiments and evaluations were carried out in the same manner as in Example 1 except that 1-heptene was changed to 1-dodecene.

Table 1 shows the dry weights of the cells and polymers obtained in Examples 1 to 6.

Example Cell dry weight (mg) Polymer dry weight (mg) One 160 48 2 170 52 3 160 55 4 180 58 5 170 55 6 160 48

(Analysis and evaluation)

The unit analysis of the polymer obtained in Examples 1-6 was performed as follows. That is, about 10 mg of PHA was placed in a 25 mL volume-type flask, dissolved in 2 mL of chloroform, 2 mL of a methanol solution containing 3% sulfuric acid was added, and the mixture was reacted at reflux at 100 ° C. for 3.5 hours. After completion of the reaction, 10 mL of deionized water was added, the resulting mixture was vibrated vigorously for 10 minutes, and the lower chloroform layer separated into two layers was taken out, dehydrated with magnesium sulfate, and the chloroform layer was subjected to gas chromatography-mass spectrometry. The methyl esterified product of the PHA monomer unit was confirmed on the apparatus (GC-MS, Shimadzu QP-5050 model, EI method). Table 2 shows the results of the area% of the total ion chromatogram (TIC). In this case, the epoxy unit could not be detected because the monomer unit was converted by methanolysis.

Example Unit One 2 3 4 5 6 C4 0.5 - - - - - C5 2.0 - - - - - C6 0.7 5.3 1.3 3.0 1.4 2.2 C6 = - 0.9 - 1.3 - 0.7 C7 7.8 12.7 6.9 4.5 3.9 2.5 C7 = 87.2 2.2 5.2 1.3 2.3 - C8 - 29.4 17.3 13.3 8.7 9.8 C8 = - 38.5 - 28.1 12.8 19.4 C9 - - 24.5 9.7 12.5 5.7 C9 = - - 43.6 - 15.8 - C10 1.8 5.4 1.2 11.4 10.6 10.3 C10 = - - - 27.4 18.5 24.0 C11 - - - - 3.6 3.5 C11 = - - - - 9.9 - C12 - 1.9 - - - 5.7 C12 = - 3.5 - - - 15.3

In Table 2, the symbols used to denote units have the following meanings.

C4: 3-hydroxybutyric acid; C5: 3-hydroxygilacetic acid; C6: 3-hydroxyhexanoic acid;

C6 =: 3-hydroxy-5-hexenoic acid; C7: 3-hydroxyheptanoic acid;

C7 =: 3-sidoxy-6-heptenic acid; C8: 3-hydroxyoctanoic acid;

C8 =: 3-hydroxy-7-octenic acid; C9: 3-hydroxynonanoic acid;

C9 =: 3-hydroxy-8-nonenoic acid; C10: 3-hydroxydecanoic acid;

C10 =: 3-hydroxy-9-decenoic acid; C11: 3-hydroxyundecanoic acid;

C11 =: 3-hydroxy-10-undecanoic acid; C12: 3-hydroxydodecanoic acid;

C12 =: 3-hydroxy-11-dodecanoic acid.

The polymers obtained in Examples 1 to 6 were subjected to 1 H-NMR analysis (device used: FT-NMR: Bruker DPX400; measuring nucleus: 1H; solvent used: medium chloroform (with TMS). Proton binding to terminal double bonds and epoxy groups was performed according to Macromolecules, 31, P1480-1486 (1998) The obtained spectra are shown in Figs.

Table 3 shows mol% of each side chain unit (terminal saturation, terminal unsaturated (double bond), terminal epoxy) calculated from these.

Carbon source Monomer Unit (mol%) Saturator A terminal unsaturated group Other epoxidizers Unsaturated group Hexene 70.0 20.0 ND ^** 10.0 Heptene 12.5 83.3 4.2 ND Octene 55.9 29.4 14.7 ND Nonen 44.0 40.0 16.0 ND Dessen 31.6 52.6 15.8 ND Undesen 30.0 50.0 20.0 ND Dodeden 39.1 43.5 17.4 ND

Note: * mol% of monomer unit was confirmed by integration with 1H-NMR.

** ND means "not detected".

In addition, the molecular weights of the polymers obtained in Examples 1 to 6 were evaluated by GPC (Toso Corporation HLC-8020; Column: Polymer Lab, PL gel MIXED-c (5 µm); Solvent: Chloroform; Converted based on polystyrene). . The results are shown in Table 4.

Example Number average molecular weight (Mn) × 10 ⁵ Number average molecular weight (Mw) × 10 ⁵ One 1.9 5.2 2 2.5 5.5 3 2.6 5.3 4 1.9 5.4 5 1.9 5.4 6 2.0 4.9

(Example 7)

YN2 strains were incubated for 24 hours at 30 DEG C in a medium containing 0.5% of polypeptone, collected by centrifugation, and resuspended in an inorganic salt medium. 10 mL of this suspension was transferred to a 27 mL volume vial, sealed with a butyl rubber flop and an aluminum chamber, and air containing 1-hexene gas was added to the syringe. As a control, samples of only inorganic salt medium containing no YN2 strain were similarly prepared, and each vial bottle was vibrated at 30 ° C for 1 hour. After the vibration, 0.1 mL of the gas phase portion of the vial bottle was collected with a syringe, and subjected to gas chromatography (GC) analysis. The conditions of GC are as follows.

Analyzer: Shimadzu GC-14B; Column: DB-624 from J &W; Column temperature: 100 ° C. constant temperature; Injector / detector temperature: 230 ° C .; detector: FID

The results are shown in Figs. 8 (a) to 8 (c). Fig. 8 (a) shows the results of samples of only inorganic salt medium containing no YN2 strain. A peak of 1-hexene can be seen around 1.05 minutes. Figure 8 (b) shows the results of the sample suspension of YN2 strain. The peak which was not seen in FIG. 8 (a) appears around 2.47 minutes. 8 (c) shows the results of the standard samples of 1,2-epoxyhexane. The peak corresponding to the said peak can be seen in the vicinity of 2.47 minutes. From the above result, it became clear that YN2 strain is converting 1-hexene into 1,2-epoxyhexane.

(Example 8)

The conversion behavior of YN2 strains for 1-octene was evaluated in the same manner as in Example 7 (GC column temperature: 150 ° C). The results are shown in Figs. 9 (a) to 9 (c). 9 (a) shows the results of a sample of only the inorganic salt medium containing no YN2 strain. The peak of 1-octene can be seen around 1.21 minutes. Figure 9 (b) shows the results of the sample suspension of YN2 strain. The peak which was not seen in FIG. 9 (a) appears around 2.38 minutes. 9 (c) shows the result of the standard sample of 1,2-epoxyoctane. The peak corresponding to the said peak can be seen in the vicinity of 2.38 minutes. From the above result, it became clear that YN2 strain is converting 1-octene into 1,2-epoxyoctane.

That is, it became clear from the result of Example 7, 8 that YN2 strain has the ability to epoxidize 1-alkene to the corresponding 1,2-epoxyalkane.

(Example 9)

20 mg of the polymer obtained in Example 4 was dissolved in 0.2 mL of chloroform, and 10 mg of hexamethylenediamine was added and dissolved while cooling with ice. Chloroform was removed after confirming completion of the dissolution, and the resulting solution was measured by a differential scanning calorimeter (DSC; Perkin Elmer, Pyris 1; elevated temperature: 10 ° C / min). Moreover, DSC measurement was performed similarly to what made the other sample react at 90 degreeC for 1 hour.

The results are shown in FIG. In the drawing, the chart indicated by (1) is a sample of the former (mixing only), and the chart indicated by (2) is the latter (reacted at 90 ° C for 1 hour). In the chart (1), a clear exothermic peak can be seen around 90 ° C., and the reaction between the epoxy group in the polymer obtained in Example 4 and the hexamethylenediamine occurs, and the crosslinking between the polymers is in progress. On the other hand, clear heat flow is not seen in the chart 2, and it shows that crosslinking reaction is completed.

In addition, infrared absorption was measured for the same sample (FT-IR; Perkin Elmer, 1720X model). The results are shown in Figs. 11 (a) and 11 (b). The peaks of Figure 11 (a) see Chart amine (3340㎝ ^-1 vicinity) could in and an epoxy group (near 822㎝ ^-1) are no longer observed in the chart of Figure 11 (b).

From the above results, the polyether having an epoxy unit in the side chain obtained by a method having a step of contacting the 1-alkene with a microorganism having the ability to introduce the 1-alkene and converting it into the polyester and converting the polyester into this polyester It has become apparent that a crosslinked polymer is obtained by reacting an ester with hexamethylenediamine.

Production of a polyester comprising 3-hydroxyfatty acid having an epoxy unit in the side chain with 1-alkene having 7 to 12 carbon atoms as a single carbon source by the method of the present invention, and the preparation of a crosslinked polymer from the obtained polymer It became possible.

Claims (16)

A method for producing a polyester containing an epoxy group in a side chain using 1-alkene as a raw material, wherein the 1-alkene is brought into contact with a microorganism having the ability to introduce the 1-alkene and convert it to the polyester. A process for producing a polyester comprising an epoxy group in a side chain, comprising a step of converting an alkene into a polyester containing an epoxy group in a side chain by the microorganism. The method of claim 1 wherein the microorganism is (a) the ability to epoxidize 1-alkenes into epoxy alkanes; (b) the ability to convert this epoxy alkane compound into epoxidized carboxylic acid, and (c) the ability to convert this epoxidized carboxylic acid to this polyester, Way with. The method of claim 1, further comprising culturing the microorganism in a medium comprising 1-alkene. 4. The method of claim 3, further comprising the step of isolating this polyester produced by the microorganism. The method according to claim 4, wherein the isolation step is a step of recovering this polyester from the microbial cells. The method of claim 1 wherein the 1-alkene is 1-alkene having from 7 to 12 carbon atoms. The process of claim 1 wherein the polyester is formulated into a monomer unit: (n: integer of 1 to 7) At least 1 mol% of the units represented by the formula. 8. A process according to claim 7, wherein the polyester is incorporated into the monomer unit. (m: integer of 1 to 7) At least 1 mol% of the units represented by the formula. 8. A process according to claim 7, wherein the polyester is incorporated into the monomer unit. (k: integer from 0 to 8) At least 1 mol% of the units represented by the formula. The method according to claim 1, wherein the number average molecular weight of the polyester is 10,000 to 1,000,000. The method of claim 1, wherein the microorganism is a microorganism belonging to Pseudomonas sp. 12. The method of claim 11, wherein the microorganism is Pseudomonas cichorii YN2 (FERM BP-7375). A method for producing a crosslinked polymer comprising reacting a polyester obtained by the method according to claim 1 with a diamine compound. The method according to claim 13, wherein the diamine compound is hexamethylenediamine. The method of claim 13, wherein the reaction is performed at a temperature in the range of 50 ° C. to 120 ° C. 15. The method of claim 13, wherein said reaction is conducted for 10 to 120 minutes.